Approximately 30 million cells are collected in a sterile 50-ml tube (Falcon type) for each extraction and pelleted by centrifugation (3000 g, 5 min, 4°). Jean-Pierre Jacquot, in Methods in Enzymology, 2002 Northern Blots Then isopropanol (added to the aqueous phase) causes the RNA to precipitate out. Guanidinium cyanate aids in the denaturation of proteins (including RNase) and β-mercaptoethanol, and sarcosine too acts as denaturing agents. In neutral condition both RNA and DNA partitioned in the aqueous phase. The acidic pH provided by sodium acetate causes the DNA to be organic while RNA at the aqueous phase.

This results in the upper aqueous phase containing nucleic acid and the lower organic phase have protein. The basis of the method is phase separation on the centrifugation of a mixture of aqueous sample and solution of phenol and chloroform. Therefore, many protocols of RNA extraction include the usage of strong denaturants as guanidium salt, which disrupts the cells, solubilizes its components, and denature the endogenous RNase at the same time. Shweta Pandey, in Protocols in Biochemistry and Clinical Biochemistry, 2021 Rationaleĭuring RNA extraction, the most important step is the inactivation of endogenous RNase.

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